Characterization of the Microbiota in the Crop of the Leech

The early bacteriological investigations in the 1940s and 1950s described the isolation of a single bacterium from the crop of the medicinal leech (Lehmensick 1941; Hornbostel 1942; Busing 1951; Busing et al. 1953). The consistent isolation of this bacterium and the absence of other microorganisms lead the early investigators to suggest a symbiotic association. Three functions have been proposed for the symbiont (Busing et al. 1953; Graf 2000, 2002): (1) digestion of the ingested blood meal, (2) synthesis of essential nutrients such as vitamin B for the host and (3) preventing the growth of other microorganisms in the crop of the leech. To the knowledge of this author, the only experimental evidence for an influence of the symbionts on host physiology comes from two studies (Busing et al. 1953; Zebe et al. 1986) that used relatively high antibiotic concentrations such that a direct effect on the host physiology could not be excluded (Graf 2002). These studies indicated that release of nitrogenous metabolic end products from the animal and the consumption of oxygen by the animal were affected by antibiotic treatment, thereby suggesting a role of the microbial partners. Further studies are needed to determine the role of the symbionts, but the ability of the leech to survive for nine months without feeding makes the determination of the contribution by the symbiont more difficult.

As the taxonomy of bacteria improved, the symbiont was renamed A. hydrophila (Jennings and van der Lande 1967). Similarly, the bacteria isolated from leech therapy associated wound infections were identified as A. hydrophila (Lowen et al. 1989; Snower et al. 1989; Lineaweaver 1991). The taxonomy Aeromonas has become more sophisticated and with it the complexity of identifying species (Janda and Abbott 1998). In one of our studies, we identified symbionts from animals from various sources using biochemical tests and 16S rRNA gene sequences and detected only A. veronii biovar sobria (Graf 1999). However, some of our strains that were isolated from leeches differed in key biochemical tests from the type strain. These differences may have resulted in the misidentification of these strains using commercial test kits. Other studies also reported the presence of various other bacteria in the digestive tract of the medicinal leech in addition to Aeromonas (Nonomura et al. 1996; Mackay et al. 1999; Eroglu et al. 2001). In these studies, it is unclear what proportion of the isolates from one animal belongs to which species. This lack of quantitative data does not allow one to determine whether these bacteria are always part of the digestive tract community, whether they represent contaminations from other organs, were introduced with a contaminated blood meal or are passing transiently through the digestive tract.

An open question is the presence of bacteria in the crop that do not grow when standard culturing techniques are used. We are currently addressing this question by cloning and sequencing 16S rRNA genes from the crop and intestinum. The initial results suggest the presence of up to three species in the crop of which two species, A. veronii biovar sobria and a species belonging to the Bacteroidetes group, appear to be the dominant players (Worthen, P., C. Gode and J. Graf, unpubl.). These results indicate that the microbial community is more complex than we had anticipated but sufficiently simple to allow us to investigate the population dynamics and molecular requirements underlying this symbiosis. Indeed, it will be important to understand microbial consortia because monospecific associations represent the exception and simple microbial consortia are likely to reveal mechanisms that are applicable to more complex associations.

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